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SummaryOrientation of the cell division axis is essential for the correctdevelopment and maintenance of tissue morphology, both forsymmetric cell divisions and for the asymmetric distribution of fatedeterminants during, for example, stem cell divisions. Oriented celldivision depends on the positioning of the mitotic spindle relativeto an axis of polarity. Recent studies have illuminated anexpanding list of spindle orientation regulators, and a molecularmodel for how cells couple cortical polarity with spindlepositioning has begun to emerge. Here, we review both the well-established spindle orientation pathways and recently identifiedregulators, focusing on how communication between the cellcortex and the spindle is achieved, to provide a contemporaryview of how positioning of the mitotic spindle occurs.

Key words: Spindle, Microtubules, Oriented cell division, Polarity,Mitosis, Centrosome

IntroductionAll multicellular animals are tasked with two fundamentaldevelopmental challenges: generating cellular diversity and formingthree-dimensional tissues, both of which initiate from a single-celled zygote. Cellular diversity is spawned by cell divisionsyielding non-identical daughters, and tissue morphogenesis isestablished through the precise three-dimensional arrangement ofcell divisions that form the overall architecture of the organism.Both of these essential challenges are resolved in part throughoriented cell division, which regulates embryogenesis,organogenesis and cellular differentiation. Notably, oriented celldivisions, and hence asymmetric cell divisions, remain crucialthroughout adulthood as well, functioning as the basis for tissuehomeostasis during growth and wound repair. One primary featureof oriented cell division is the proper positioning of the mitoticspindle relative to a defined polarity axis. In principle, spindleorientation is achieved through signaling pathways that provide amolecular link between the cell cortex and spindle microtubules.These pathways are thought to elicit both static connections anddynamic forces on the spindle to achieve the desired orientationprior to cell division. Although our knowledge of the signalingmolecules involved in this process and our understanding of howthey each function at the molecular level remain limited, collectiveefforts over the years have shed light on the importance of spindleorientation to animal development and function. Moreover,emerging evidence shows an association between improper spindleorientation and a number of developmental diseases as well astumor formation. The study of spindle orientation is thereforefundamental to both developmental biology and human disease.

Over a century ago, Oscar Hertwig discovered that sea urchinembryos biased the orientation of the mitotic spindle along their longaxis, which led to a model (the ‘Hertwig Rule’) in which cells orientdivisions in response to mechanical forces (Hertwig, 1884). TheHertwig model stated that mechanical regulation was the primarydeterminant of spindle orientation as cells sensed shape changes inresponse to external forces. Later discoveries, including those inascidian embryos, in which cell division orientation correlated withdifferential daughter cell fate and size, suggested a molecular basisfor spindle orientation (Conklin, 1905). Conklin reasoned that theability to alter division orientation to achieve autonomous fatespecification during development must rely upon internal molecularregulation rather than external mechanics. The modern molecular erahas now vastly expanded our understanding of oriented cell division,with studies showing support for both models.

Genetic identification of the first spindle orientation regulatorsoccurred nearly two decades ago (Cheng et al., 1995; Etemad-Moghadam et al., 1995; Kraut et al., 1996; Zwaal et al., 1996).Since then, the ‘parts list’ of proteins required for proper spindleorientation has grown tremendously, and includes componentsfrom several signaling pathways that couple the mitotic spindle tocortical polarity complexes in a variety of cell types from a diversegroup of organisms. More recent studies utilizing genetic screeningtechnologies and improved cell culture-based systems haveexpanded that list further. Moreover, these recent studies havebegun to view spindle orientation through a more molecularlyfocused lens that allows better insight into how these parts fittogether during oriented cell divisions.

The ability to bias the orientation of cell division via regulatedspindle positioning is conserved from yeast to mammals (Siller andDoe, 2009). For the scope of this Review, we have chosen to focuson selective, well-studied examples from metazoan modelorganisms. We first highlight several developmental processes thatrely on regulated spindle orientation/asymmetric cell divisions andprovide an overview of how cell polarity, and hence spindleorientation, is first established in these examples. A particular focuswill be placed on the mechanisms by which cortical polarity cues‘capture’ spindle microtubules, as this process represents an earlystep in the spindle orientation process. Although microtubule captureitself is likely to be a conserved and generalized aspect of spindleorientation, the underlying molecular pathways appear to be diverse,highlighting the importance of further understanding of themechanisms involved. We describe several well-established spindleorientation components as well as newly identified factors thatregulate communication between the cell cortex and the mitoticspindle, with an aim to provide readers with an updated view of howdifferent cell types regulate the position of the mitotic spindle.

Oriented/asymmetric cell divisions duringmetazoan developmentIt should be noted that oriented/asymmetric cell division, asdiscussed throughout this Review, is defined by two mutually

Development 140, 1843-1856 (2013) doi:10.1242/dev.087627© 2013. Published by The Company of Biologists Ltd

Molecular pathways regulating mitotic spindle orientation inanimal cellsMichelle S. Lu1 and Christopher A. Johnston2,*

1Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA.2Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA.

*Author for correspondence (johnstca@unm.edu)

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coupled events: (1) establishment of a cortical polarity axis by theunequal distribution of polarity proteins and cell fatedeterminants; and (2) alignment of the mitotic spindle withrespect to this polarity axis. In certain cases, the polarity axiscoincides with a tissue or body axis of the organism. We willfocus our discussion primarily on how these two processes arelinked at the molecular level using four examples: theCaenorhabditis elegans zygote, Drosophila neuroblasts,Drosophila sensory organ precursors and mammalian epidermalcells. As gene and protein names often vary between species,please see Table 1 for the naming of orthologs.

The first series of divisions in the C. elegans zygote producessiblings that are asymmetric in both size and fate (Fig. 1A). Earlygenetic studies revealed a set of evolutionarily conservedpartitioning-defective (PAR) proteins that are necessary forestablishing an anterior-posterior (A-P) cortical polarity axis priorto the first zygotic division (Kemphues et al., 1988). Subsequentstudies demonstrated that the PAR complex also regulates spindleorientation and the unequal force generation exerted on the spindlepoles (Etemad-Moghadam et al., 1995; Grill et al., 2001;Kemphues et al., 1988). Defects in PAR complex genes result inmispositioning of the mitotic spindle, loss of daughter cellasymmetry and, ultimately, non-viable animals. As shown inFig. 1A, two successive divisions with A-P spindle orientationsproduce a four-cell embryo containing a blastomere known as theEMS blastomere. Spindle orientation along the A-P axis andsubsequent asymmetric cell division of the EMS cell then generatesan E daughter cell, which will give rise to endodermal lineages, anda MS daughter cell, which will form mesodermal lineages. Spindleorientation in the one-cell stage and the P2 blastomere cell requiresthe activity of GPR-1/2 and LIN-5, which constitute anevolutionarily conserved non-canonical G-protein signalingnetwork (Colombo et al., 2003; Gönczy, 2008; Werts et al., 2011).Mutations in Wnt signaling pathway components result inmisalignment of the EMS spindle and mis-specification of germcell layers (Schlesinger et al., 1999; Walston et al., 2004) (Fig. 1A).By manipulating contact sites at the four-cell stage, Goldsteinshowed that cell-cell contacts establish a site that capturescentrosomes via emanating microtubules to orient cell divisions(Goldstein, 1995). Actin-rich contact sites between the EMS andP2 cells determined spindle orientation and influenced partitioningof fate information necessary for gut specification (Goldstein,1995; Waddle et al., 1994). Collectively, these studies suggest thatspindle orientation is an essential determinant of cell fatespecification during C. elegans development.

Asymmetric division of Drosophila neuroblasts, the stem cellsof the developing fly brain, regulates development of the fly centralnervous system (Fig. 1B). Neuroblasts polarize along an apical-basal (A-B) axis and divide in a stem cell-like manner to producea self-renewed neuroblast and a ganglion mother cell (GMC) thatproduces differentiated neurons and glia (Doe, 2008). Thus, arelatively small number of neuroblasts can supply the vast numberof differentiated neuronal cells that constitute the adult brain.Genetic studies over the past decade have identified three coreprotein complexes (Fig. 1B) that ensure proper asymmetricneuroblast division: (1) the apical ‘polarity complex’ consisting ofthe evolutionarily conserved proteins Par-3 (also known asBazooka), Par-6 and atypical protein kinase C (aPKC) (Petronczkiand Knoblich, 2001; Wodarz et al., 1999); (2) the apical ‘spindleorientation complex’ consisting of Inscuteable (Insc), Partner ofInscuteable (Pins; also known as Rapsynoid) and Mushroom bodydefect (Mud) (Schaefer et al., 2000; Schober et al., 1999; Siller et

al., 2006; Yu et al., 2000); and (3) the basal ‘differentiationcomplex’ consisting of the adapter protein Miranda (Mira) and cellfate markers such as Prospero (Pros), Brain tumor (Brat) and Numb(Betschinger et al., 2006; Lee et al., 2006b). Proper A-B spindlepositioning ensures apical inheritance of aPKC, which promotesself-renewal, and basal inheritance of the Miranda complex, whichinduces neuronal differentiation (Fig. 1B). Defects in these corecomponents uncouple spindle orientation from the polarity axis,disrupting division asymmetry and often resulting inoverproliferation of neural stem cells at the expense ofdifferentiated progeny. This dysregulated division pattern can resultin neuroblast-based tumors, loss of neuronal production, andlethality (Cabernard and Doe, 2009; Lee et al., 2006b) (see Box 1).Thus, spindle orientation with respect to intrinsic polarity cuesensures proper stem cell homeostasis during development.

Drosophila sensory organ precursors (SOPs) are ectodermalprogenitor cells that give rise to mechanosensory organs of thefly peripheral nervous system (Fig. 1C). Each SOP in theimaginal disc of the developing wing undergoes a series ofdivisions, the orientation of which is crucial for both asymmetricsibling fate specification and production of the proper structuralintegrity and alignment of individual sensory wing hairs(Fig. 1C). Cortical spindle orientation cues in SOPs localizethrough an evolutionarily conserved mechanism known as planar

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Box 1. Oriented/asymmetric cell division and diseaseDefects in spindle orientation are associated with malignantneuroblast-based tumors caused by uncontrolled self-renewaldivisions in Drosophila (Gonzalez, 2007; Lee et al., 2006b). Severalprominent tumor suppressor proteins, including APC, Dlg, VHL(Thoma et al., 2010; Thoma et al., 2009) and LKB1, regulate spindleorientation (Table 1), suggesting that spindle misorientation mightcontribute to tumor development (Pease and Tirnauer, 2011).

Neurogenesis requires control of cell division orientation inneuroprogenitors at specific developmental stages (Lancaster andKnoblich, 2012) to balance proliferative and neurogenic outcomes(Konno et al., 2008; Peyre et al., 2011). Mutations in the spindleorientation regulators LIS1 (PAFAH1B1) and HTT manifest in Type Ilissencephaly and Huntington’s disease, respectively. Interestingly,both LIS1 and HTT regulate the function of cytoplasmic dynein, apotentially universal regulator of spindle orientation (Table 1; seemain text). Whether a causative molecular link exists betweenspindle orientation and neurodevelopmental disorders will be animportant future research endeavor.

Components of the intraflagellar transport (IFT) machinery in ciliahave been linked to misoriented cell divisions possibly underlyingpolycystic kidney disease (PKD) (Delaval et al., 2011; Fischer et al.,2006; Hildebrandt and Otto, 2005). Several IFT proteins localize tocentrosomes, and mutations in IFT88 result in spindle misorientation(Delaval et al., 2011), leading to improper spatial arrangement ofnephron epithelia and cyst development. Interestingly, IFT88participates in astral microtubule formation possibly through adynein-dependent transport complex (Delaval et al., 2011).However, mutations in other IFT components appear to drivecystogenesis through mechanisms independent of spindlemisorientation (Jonassen et al., 2012).

Intestinal crypt stem cells regulate spindle orientation to producedifferentiated progeny. Heterozygous mutations in the tumorsuppressor protein APC, which is mutated in ~85% of colorectalcancer cases (Markowitz and Bertagnolli, 2009), results in spindlemisorientation and altered cell shape (Quyn et al., 2010). However,this dependence of intestinal stem cells on divisionorientation/asymmetric division has been challenged recently andremains controversial (Schepers et al., 2011).

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Table 1. Identified regulators of spindle orientation

Protein name Proposed localization and function References

Dm/Hs: Inscuteable Cortically polarized. Recruits and stabilizes Pins at the cortex. Du et al., 2001; Kraut et al., 1996; Schaefer et al., 2000; Schober et al., 1999; Woodarz et al., 1999

Dm: Pins (Raps) Ce: GPR-1/2 Hs: LGN (GPSM2)

Cortically polarized. Recruits Mud-Dynein and Dlg-Khc-73 complexes to connect to spindle MTs.

Colombo et al., 2003; Du et al., 2001; Johnston et al., 2009; Lechler and Fuchs, 2005; Schaefer et al., 2000

Dm: Mud Ce: LIN-5 Hs: NuMA

(NUMA1)

Cortically polarized, centrosomes, spindle MTs. Binds Pins TPR domains; associates with Dynein-Dynactin complex; participates in cortical force generation on the spindle.

Bowman et al., 2006; Colombo et al., 2003; Du and Macara, 2004; Gönczy et al., 1999; Izumi et al., 2006; Johnston et al., 2009; Siller et al., 2006; Siller and Doe, 2008

Dm/Ce/Hs: cytoplasmic dynein

Spindle MTs. Minus-end-directed motor protein; transduction of cortical pulling forces.

Gönczy et al., 1999; Kotak et al., 2012; Johnston et al., 2009; Siller and Doe, 2008

Dm: CLIP-190 Hs: CLIP-170 (CLIP1)

CAP-Gly domain, MT plus-end binding. Promotes MT rescue from catastrophe, resulting in MT growth towards cortex; yeast homolog may activate dynein.

Akhmanova et al., 2001; Komarova et al., 2002; Miller et al., 2006; Sheeman et al., 2003

Dm: Glued Ce: DNC-1 Hs: p150-Dynactin

(DCTN1)

CAP-Gly domain, MT plus-end binding. Required for centrosome orientation in C. elegans; Glued regulates spindle assembly; enhances Dynein processivity.

Culver-Hanlon et al., 2006; Gönczy et al., 1999; Siller et al., 2005

Dm: Ctp Ce/Hs: DLC

Spindle MTs and centrosome. Component of the Dynein complex; binds the centrosomal protein Ana2; promotes cortical Pins-Mud complex.

Wang et al., 2011

Dm: Dlg (Dlg1) Apical cortex enrichment in neuroblasts. Binds phosphorylated Pins; associates with Khc-73; provides ‘MT capture’ pathway at cortex.

Johnston et al., 2009; Johnston et al., 2012; Siegrist and Doe, 2005

Dm: Khc-73 MT plus-ends. Plus-end tracking MT motor; associates with Pins-Dlg complex; provides static link from cortex to spindle MT plus-ends.

Hanada (2000); Johnston et al., 2009; Johnston et al., 2012; Siegrist and Doe, 2005

Dm: Dsh Ce: DSH Dr: Dvl

Cortically polarized. Functions downstream of Wnt/Fz morphogenic signaling; binds Mud-Dynein complex similar to Pins; regulates function of APC; may involve additional non-canonical pathways (e.g. activation of Rho superfamily GTPase).

Schlesinger et al., 1999; Ségalen et al., 2010; Walston et al., 2004

Dm: LIS-1 Probably spindle-associated. Associates with and regulates Dynein function; participates in MT stabilization; stimulates Dynein-dependent spindle movements.

Johnston et al., 2009; Siller and Doe, 2008

Dm: Htt Hs: HTT

Spindle poles. Regulates localization of Dynein, Dynactin and NuMA. Godin et al., 2010

Dm/Hs: VHL Spindle microtubules. Loss of VHL leads to unstable astral MTs and loss of spindle checkpoint; inhibits MT catastrophe and promotes rescue frequency.

Thoma et al., 2009; Thoma et al., 2010

Dm: EB1 MT plus-ends. Regulates MT assembly, stability and dynamics; essential for plus-end trafficking of many other components.

Rogers et al., 2002; Toyoshima and Nishida, 2007; Wen et al., 2004

Dm/Ce: APC MT plus-ends. Stabilizes spindle MTs; functions upstream of Rac in C. elegans gonad development; orients the mother centrosome in mGSCs; controls planar orientation of intestinal epithelial cells.

Cabello et al., 2010; Fleming et al., 2009; Walston et al., 2004; Wen et al., 2004; Yamashita et al., 2003

Dm: Akt (Akt1) Cell cortex. Regulates centrosome migration; promotes proper spindle morphology; necessary for cortical APC localization.

Buttrick et al., 2008

Dm: Par-1 Ce: PAR-1 Hs: MARK1

Putative EB1 interaction (MT plus-ends); cell cortex in C. elegans embryos. Kinase activity helps establish polarity of other components; ensures centrosome orientation in Drosophila mGSCs.

Tabler et al., 2010; Wu and Rose, 2007; Yuan et al., 2012

Hs: MCAK (Kinesin-13; KIF2C)

Putative EB1 interaction (MT plus-ends). MT depolymerase, promotes catastrophe; activity is coupled to spindle force generation.

Grill et al., 2001; Tanenbaum et al., 2011

Xl/Hs: PAK Cytoplasmic. Phosphorylates Ran to promote activation at centrosome; Ran promotes Mud localization.

Bompard et al., 2010; Speicher et al., 2008; Wee et al., 2011

Dm: Canoe Hs: Afadin

MLLT4

Cortical polarized in Drosophila neuroblasts; component of epithelial junctions. Binds directly to Pins; recruits activated Ran and promotes cortical Mud localization.

Speicher et al., 2008; Wee et al., 2011

Dm: Aurora Hs: Aurora-A

(aurora kinase A)

Spindle poles. Kinase activated in mitosis; phosphorylates Pins to promote Dlg-Khc-73 pathway activation; also promotes proper cortical polarity.

Johnston et al., 2009; Lee et al., 2006a

Dm: (SNF1A) Hs: AMPK (PRKAA1)

Spindle poles. Kinase that promotes astral MT organization; Myosin light chain is putative effector; requires phosphorylation by LKB1 to become activated.

Thaiparambil et al., 2012

Ce: (CSK-1) Dm: Dsrc41 Hs: c-Src (CSK)

Spindle poles. Important for astral MT organization and spindle pole development; may act downstream of Wnt signaling; target of phosphorylation currently unknown.

Bei et al., 2002; Nakayama et al., 2012

Hs: ABL1 Cytoplasmic. Phosphorylates LGN to promote its polarity; phosphorylates NuMA to promote maintenance of LGN-NuMA complex.

Matsumura et al., 2012

Ce: PKC-3 Dm: aPKC Hs: PRKZ

Cortically polarized. Phosphorylates LGN, promotes 14-3-3 binding and cortical removal; regulaties LGN polarity in epithelial cells.

Hao et al., 2010

Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Dr, Danio rerio; Hs, Homo sapiens; MTs, microtubules; Xl, Xenopus laevis. Although Hs proteins are listed, in some cases other mammalian systems were used in the studies mentioned. D

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cell polarity (PCP) within the imaginal disc tissue. The spindlemust be controlled in both an A-B orientation, through the actionof the Pins complex, as well as an A-P orientation, which iscontrolled by a non-canonical Frizzled/Dishevelled (Fz/Dsh)signaling pathway (David et al., 2005; Gho and Schweisguth,1998; Roegiers et al., 2001) (Fig. 1C). Proper asymmetricdivision in SOPs further relies on the activity of core PCPcomponents [a discussion of which is beyond the scope of thisReview, but see Wallingford (Wallingford, 2012) for an excellent

recent review]. The disruption of spindle orientation duringasymmetric SOP divisions results in mis-specification of celllineages and defects in bristle development (Bellaïche et al.,2001; Lu et al., 1999). Thus, regulated spindle orientation playsa central role in the development of multicellular sensorystructures.

Basal epidermal cells contribute to the architecture of theepidermis, a stratified squamous epithelium of skin that regulatesfluid and electrolyte exchange and guards against harmful or

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B D. melanogaster neuroblast

A C. elegans embryo

C D. melanogaster sensory organ precursor

D M. musculus epidermis

Par-3, Par-6, aPKC, Insc

PAR-3, PAR-6, PKC-3

GPR-1/2, LIN-5

PAR-2, PAR-1

Pins

Mud

Fz-Dsh

h

pIpIIb pIIa

sh

gn

so

LGN/NuMA

Notch

Unpolarized LGN/NuMA

sb

ep

Fz-Dsh

GMC

NBPins, Dlg, Mud, Khc-73

Mira, Pros, Brat

Early Late

A P

B

A

sb

A P

B

A

B

A

A P

Key

Key

Key

Key

EMS

P2

Fig. 1. Spindle positioning regulates oriented/asymmetric cell division during metazoan development. (A) Spindle orientation regulatesasymmetric cell divisions in early C. elegans development. The first zygotic division produces daughter cells that are asymmetric in both size and fatespecification. Prior to division, the cell cortex is polarized along an anterior-posterior (A-P) axis by the activity of PAR (blue) and PKC-3 (red). Spindleorientation is coupled to this polarity axis primarily through the action of the GPR-1/2 and LIN-5 complex (yellow/green), which enriches along theposterior cortex. In subsequent divisions, the Fz-Dsh complex (orange) regulates spindle orientation in the EMS cell. Similar to the one-cell stage, PARcomplexes show reciprocal polarity in EMS and P2 cells (Arata et al., 2010), whereas GPR-1/2 influences spindle positioning through asymmetriclocalization in the P2 cell specifically (Werts, Roh-Johnson and Goldstein, 2011). (B) Drosophila neuroblasts polarize along an apical-basal (A-B) axisthrough the activity of the Par-aPKC complex (red). Spindle orientation is regulated by the apical Pins complex, which recruits the regulatory proteinsMud, Dlg and Khc-73 (yellow/green). Neuroblast asymmetric divisions result in a larger self-renewed neuroblast (NB) and a smaller ganglion mother cell(GMC), which is specified for neuronal differentiation by the inheritance of the Mira-Pros-Brat complex (blue). (C) Drosophila sensory organ precursorcells (SOPs) in the epithelium of developing wing imaginal discs give rise to the adult mechanosensory bristles. Each SOP progenitor gives rise to fivedistinct cells that constitute the entire bristle structure: g, glial; h, hair; n, neuron; sh, shaft; so, socket. Spindle orientation in the initial (pI) cell division isregulated by the coordinated action of Pins (green), which positions the spindle within the epithelial plane, and Fz-Dsh (orange), which regulatesorientation along the A-P axis. Pins-mediated rotation of the spindle in the pIIb cell then establishes the A-B division orientation necessary for correctspecification and positioning of the neuronal and glial cells. (D) In the mouse skin, basal epidermal cells (ep) divide within the epithelial plane early indevelopment, resulting in expansion of the tissue (left). At later developmental stages (right), division orientation switches to an A-B mode via Pins-mediated repositioning of the mitotic spindle. This mode of division positions one daughter, the suprabasal (sb) cell, below the epithelium. The sb celldifferentially inherits Notch, which specifies differentiation resulting in stratification of the skin.

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infectious substances, such as microbes. Early in development,these progenitor basal cells preferentially divide within theepithelial plane, producing two symmetric daughter basal cells toexpand the undifferentiated basal layer (Fig. 1D). However, at alater developmental stage, basal cell divisions switch to anasymmetric mode, which allows the self-renewal of a proliferativebasal cell while also producing a suprabasal cell that is committedto differentiating into the deep tissue layers forming the skin barrier(Lechler and Fuchs, 2005). Strikingly, this asymmetric divisionswitch accompanies a 90° reorientation of the mitotic spindle, fromparallel to perpendicular to the epithelial plane, which is dependenton the Par-aPKC and Pins-Insc-Mud polarity complexes (Fig. 1D)(Lechler and Fuchs, 2005; Poulson and Lechler, 2010). Spindlereorientation ensures asymmetric inheritance of Notch, whichspecifies barrier cell differentiation. Spindle misorientation mutantsthus elicit impaired stratification and defects in barrier function,leading to dehydration and death (Williams et al., 2011). Theseresults demonstrate the evolutionary conservation of couplingspindle orientation to decisions of cell fate and tissue structure, andunderscore the importance of proper cell division orientation duringdevelopment and in disease (see Box 1).

Linking the spindle to the cell cortex: the role ofmicrotubule plus-end binding proteinsAs highlighted above, correct orientation of the spindle withrespect to cell polarity is crucial for tissue development andhomeostasis. The ability to position statically the mitotic spindle,a large, otherwise dynamic cellular structure, necessitates physicalconnections between the cell cortex and the plus-ends of astralmicrotubules emanating from the spindle poles (Fig. 2). In thismanner, the polarization of cortical cue(s) affords the cell theability to position the spindle in a biased orientation via interactionsbetween these polarized proteins and microtubule plus-end bindingproteins (+TIPs). In this model, +TIPs would serve as the prey ina ‘microtubule capture’ mechanism by interacting with cortical bait.

Metazoans have evolved a diverse set of +TIPs (Fig. 2A) withvaried domain architectures, protein structures and biologicalfunctions (Slep, 2010). For example, members of theXMAP215/CLASP family of +TIPs contain multiple domainsknown as TOG or TOG-like domains, which demonstrate directplus-end tracking activity in reconstituted systems and bind α/β-tubulin dimers directly (Fig. 2B) (Al-Bassam et al., 2007).XMAP215 members play an essential role in interphasemicrotubule polymerization and stabilization, which is necessaryfor proper formation of the mitotic spindle (Slep, 2009; Tournebizeet al., 2000). ZYG-9, a C. elegans XMAP215 member, regulatesspindle positioning in one-cell-stage embryos (Bellanger et al.,2007). A second autonomously tracking +TIP, end-binding protein1 (EB1), serves as the cellular workhorse for plus-end proteinlocalization. EB1 itself binds microtubules through an N-terminalcalponin homology (CH) domain, an interaction that is enhancedby an acidic, EEY tripeptide motif at the extreme C-terminus(Fig. 2A,C). EB1 binding induces tubulin polymerization in vitro(Slep and Vale, 2007) and is necessary for proper assembly of themitotic spindle (Rogers et al., 2002). Depletion of EB1 inDrosophila S2 cells causes a significant reduction in microtubuledynamics by inducing extended phases of no growth or ofshrinkage, although the overall morphology of interphasemicrotubules is not disrupted. By contrast, EB1 depletion in mitoticcells induces shortened astral microtubules and fragmentedmicrotubules at prophase along with compacted spindles, detachedspindle poles and unfocused nonastral spindles at metaphase

(Rogers et al., 2002). Subsequent studies indicated that EB1stabilizes microtubules through direct interaction withAdenomatous polyposis coli (APC) and the actin polymerizingformin protein Diaphanous (Dia) (Wen et al., 2004). EB1 regulatesplanar spindle orientation in nonpolarized, cultured epithelial cellsthrough a microtubule stabilization mechanism downstream of β1-integrin adhesion. This effect also requires myosin X-dependentremodeling of the actin cytoskeleton (Toyoshima and Nishida,2007). It is also worth noting that the budding yeast homolog ofEB1, Bim1, functions in spindle orientation through a complexwith Kar9 and the myosin Myo2 in a process that involves theguidance of microtubules along actin filaments (Hwang et al.,2003; Korinek et al., 2000). Thus, XMAP215 (also known as Mspsin Drosophila) and EB1 appear to regulate spindle orientationthrough the stabilization of spindle structure (Fig. 2).

In addition to its role in the formation and stabilization of spindlemicrotubules through direct plus-end binding, EB1 recruits anexpanding list of indirect +TIPs. Several of these have been shownto regulate spindle orientation, and they fall into two groups thatact through distinct EB1-interacting motifs (Kumar and Wittmann,2012; Slep, 2010). The first group, which consists of the proteinsCLIP-170 (also known as CLIP1; CLIP-190 in Drosophila) andp150-dynactin (also known as DCTN1; Glued in Drosophila), bindhomodimeric EB1 C-termini through a conserved N-terminal CAP-Gly domain (Fig. 2A,C). CLIP-170 promotes microtubule growth,which has been shown to occur selectively towards the cell cortexand may contribute to directional migration of motile cells(Akhmanova et al., 2001; Komarova et al., 2002). The buddingyeast homolog of CLIP-170, Bik1p, regulates spindle orientation,possibly through Num1p-mediated dynein activation and/orasymmetric polarization of Kar9 (Miller et al., 2006; Sheeman etal., 2003). As clear orthologs of Num1p and Kar9 are nonexistentor as yet undiscovered, the role of CLIP-170 in spindle orientationin higher eukaryotes remains unclear. A role for p150-dynactin inspindle orientation, however, has been demonstrated. In C. eleganszygotes, p150 (DNC-1) is necessary for proper centrosome rotationalong the longitudinal axis and thus spindle orientation (Skop andWhite, 1998). A subsequent study demonstrating a more robustreduction of p150 expression revealed additional roles inpronuclear migration and centrosome separation (Gönczy et al.,1999). The Drosophila ortholog, Glued, has been shown to regulateboth spindle assembly (Siller et al., 2005) and orientation (Sillerand Doe, 2008) in larval neuroblasts. These functions of Gluedoccur via enhanced processivity of dynein, an essential spindleorientation component (discussed in detail below) (Culver-Hanlonet al., 2006). Notably, alterations in Huntingtin (Htt), the proteinmutated in the neurodegenerative Huntington’s disease, result inmislocalization of p150 and subsequent spindle misorientation. Httassociates with p150 at spindle poles during mitosis (Gauthier etal., 2004); knockdown of Htt results in a partial loss of spindle-associated p150 along with shortened spindles and unfocusedspindle poles (Godin et al., 2010). This loss of Htt-mediatedspindle orientation in vivo causes mis-specification of neuralprogenitor cells in mice, providing evidence for a potential linkbetween spindle orientation, cell fate and disease (see Box 1)(Godin et al., 2010).

Members of the second family of EB1-interacting +TIPs containthe short SxIP polypeptide motif (Fig. 2A,C). The SxIP motif bindsin a slightly bent conformation to a hydrophobic cavity andadjacent ‘polar rim’ formed at the EB1 C-terminus (Honnappa etal., 2009). This mode of EB1 interaction, via a short polypeptidemotif, allows for a vast diversity of otherwise unrelated proteins to D

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localize to microtubule plus-ends (Kumar and Wittmann, 2012).Recent evidence suggests this interaction might be regulated byphosphorylation of the SxIP motif or flanking residues, pointing toa potential for cell cycle-dependent regulation (Buey et al., 2012;Honnappa et al., 2009). As noted above, EB1 binds the tumorsuppressor protein APC, which contains a clearly defined SxIPmotif, and this interaction is required for plus-end APC localization(Fig. 2A,C) (Honnappa et al., 2009). APC has a defined spindleorientation role in several model systems. Firstly, in distal tip cells

of the C. elegans gonad, APC functions upstream of Rac-mediatedspindle orientation, which is required for proper gonadaldevelopment (Cabello et al., 2010). APC may also control thetiming of spindle rotation in the ABar, a cell present at the eight-cell stage, during C. elegans blastomere development (Walston etal., 2004). Secondly, in Drosophila, male germline stem cells(GSCs) adhere to a hub cell within the testis niche and undergoasymmetric divisions to produce a proximal self-renewed stem celland a differentiated spermatagonial cell. Local signals from the

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B

C D

A

EB1

APC

MCAK

CLIP-170

p150

15/20 aa repeats SxIPArmadillo

Coiled coilCAP-GlyCAP-Gly

CAP-Gly Coiled coil Coiled coil

CH EBH EEY

KinesinSxIP

XMAP215 TOG 2TOG 1 TOG 3 TOG 4 TOG 5

TOG 1 TOGL 1 TOGL 2 CLIP170-BDCLASP

α-tubulin

EB1

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Fig. 2. Plus-end binding proteins (+TIPs) contribute to microtubule dynamics and spindle orientation. (A) Domain architectures of various +TIPs.Arrows indicate direct interactions between +TIPs. (B) The XMAP215 and CLASP family of proteins (shown in red) are autonomously tracking +TIPs thatregulate MT dynamics and spindle assembly. Interaction with microtubule (MT) plus-ends (+) occurs through direct binding of the XMAP215-CLASPTOG domain. (C) EB1 (shown in light blue) binds MT plus-ends through an N-terminal calponin homology (CH) domain and an acidic C-terminal EEYtripeptide motif. The EB1 homology domain (EBH) of EB1 also interacts with CAP-Gly domains found in CLIP-170 and p150-dynactin, thereby inducingtheir plus-end localization. CLIP-170 and p150-dynactin regulate MT dynamics, spindle assembly, centrosome positioning, and dynein activity. EB1 alsobinds a growing number of proteins containing the SxIP polypeptide motif, including APC and Par-1. The EBH/EEY domains directly bind the SxIPsequence and mediate plus-end tracking. Several members of the SxIP-containing family have been implicated in MT stability, centrosome/spindleorientation, actin polymerization, and polarity regulation/maintenance. (D) The MT depolymerase MCAK (shown in dark blue) also tracks to plus-endsthrough an EB1/SxIP interaction. MCAK-mediated depolymerization results in MT shortening and alteration in cortex-MT architecture (downwardarrow), which may be coupled to cortical force generation, which is necessary for spindle orientation (upward arrow).

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niche induce self-renewal only in the proximal cell, thusnecessitating precise orientation of division perpendicular to theniche cell. Mutations in APC result in mispositioning of the mothercentrosome and subsequent defects in spindle orientation(Yamashita et al., 2003). Neuroepithelial cells in the developingDrosophila brain also rely on APC for proper spindle orientation;APC RNA interference (RNAi) results in altered planar spindleorientation and defects in proper symmetric divisions of these cells(Lu et al., 2001). Finally, several recent studies have demonstrateda role for APC-mediated spindle orientation in mammalian smallintestinal crypt stem cells. For example, an APC truncation mutantcauses spindle misorientation that manifests as a switch from A-Bto planar orientation (Quyn et al., 2010). Moreover, analysis ofintestinal tumors in APCmin (multiple in neoplasia; containing anonsense truncation mutation at codon 850 in the APC N-terminus)mice demonstrate more severe spindle orientation phenotypes inintestinal crypt cells than in ‘normal-appearing’ heterozygous cells(although these cells show misorientation relative to wild type)(Fleming et al., 2009), suggesting the potential role of properspindle orientation as a tumor suppressor mechanism (see Box 1).These studies highlight a key role for the tumor suppressor proteinAPC in spindle positioning and suggest that its EB-1 plus-endassociation is instrumental for promoting interactions with corticalorientation cues (Fig. 2; Table 1).

A recent proteomic study identified microtubule affinity-regulating kinase 1 (MARK1; also known as PAR-1), aserine/threonine kinase, as another SxIP-containing partner of EB1(Fig. 2A,C) (Jiang et al., 2012). In C. elegans, PAR-1 regulates thecortical polarization of factors involved in positioning of themitotic spindle. Loss of PAR-1 results in embryos that fail toundergo posterior displacement of the mitotic spindle and do notmaintain proper A-P spindle orientation (Wu and Rose, 2007). Itshould be noted, however, that whether the role of C. elegans PAR-1 in these processes occurs through regulation of microtubuledynamics per se has not been firmly established (Pellettieri andSeydoux, 2002). Studies in the Xenopus embryo found that PAR-1induces ‘vertical’ spindle orientation in the neuroepithelium, whichpromotes the generation of deep, differentiated neuronal progenythrough asymmetric division patterns (Tabler et al., 2010).Interestingly, PAR-1 has also been shown to function in centrosomeorientation checkpoint, a crucial process that ensures the properoriented division of Drosophila male GSCs (Yuan et al., 2012).Although the precise mechanism through which PAR-1 acts inthese systems remains unclear, these studies suggest that it mayachieve a similar spindle orientation outcome through diversemolecular mechanisms. Moreover, whether EB1 binding and plus-end localization is required for PAR-1 function in spindleorientation has not been examined.

Finally, the kinesin-13 MCAK (also known as KIF2C),belonging to the kinesin family of microtubule motor proteinsthat typically move toward plus-ends (Hirokawa et al., 2009),also localizes to MT plus-ends via an EB1-SxIP interaction(Fig. 2A,D) (Honnappa et al., 2009; Tanenbaum et al., 2011).MCAK functions as a potent microtubule depolymerase,promoting catastrophe events and thus regulating microtubulelength and morphology. Interestingly, a recent report using anelegant reconstitution system demonstrated that the plus-enddepolymerase activity of MCAK was coupled to local forceproduction (Oguchi et al., 2011), raising the intriguing possibilitythat depolymerases could participate in spindle force generationinvolved in spindle orientation (Fig. 2D) (Grill et al., 2001)(discussed below). This EB1-dependent localization and

depolymerase activity is conserved in invertebrates, such as withthe Drosophila ortholog, Klp10A (Mennella et al., 2005). Loss ofKlp10A exacerbates microtubule polymerization, which results inincreased density, abundance and length of spindle astralmicrotubules (Morales-Mulia and Scholey, 2005). Notably, properregulation of astral microtubules is essential for mitotic spindleorientation in Drosophila neuroblasts (Siller and Doe, 2008).Although no evidence yet exists for a direct role of Kinesin-13depolymerases in spindle orientation, the results discussed abovesuggest these microtubule enzymes, along with other relatedproteins (Loughlin et al., 2011), might serve an essentialregulatory function at the interface between plus-ends and corticalspindle orientation complexes. Future studies using highresolution, real-time imaging to examine astral MT dynamicsdirectly at the cortex during oriented cell division will beextremely valuable in understanding this process. One potentialmodel would be that shortening of astral MT ends bydepolymerase enzymes is necessary to generate gaps that corticalforce generators then close through spindle movements coupledto reorientation (Fig. 2D).

In summary, a vast number of diverse protein families localizeto plus-ends through distinct mechanisms. This network of proteincomplexes appears to regulate spindle orientation by differentmechanisms, most of which are unknown and will require furtherstudies to resolve completely. More importantly, understandinghow these varied mechanisms function together at the cortex/+TIPinterface will be an important future research focus. For example,do components of the cortical spindle orientation complex affectthe activity of MT stabilizing agents at the +TIP? Conversely, dostable MTs provide regulatory feedback for activity of corticalprotein complexes? Does the cortical actin cytoskeleton play anintervening role between the cortex and MTs, perhaps by bothmaintaining cortical polarity and interacting with spindle MTs? Theactin cytoskeleton has been shown to regulate leading edge MTturnover in migratory cells (Gupton et al., 2002). Themechanism(s) by which the actin cytoskeleton contributes tospindle orientation in animal cells remains an area of emerginginvestigation. Local changes in cortical actin polymerization,induced by the activity of +TIPs, such as APC and Dia, couldprovide a site for MT capture and spindle stabilization. In turn,stable MTs might provide a site for actin-MT crosslinking proteininteraction (Rodriguez et al., 2003). Alternatively, +TIP-inducedactin regulation could play an important role in stabilizing thecortical localization and polarity of other spindle orientationregulators (Li and Gundersen, 2008). Regulation of the corticalactin network through external forces (Fink et al., 2011) or integrin-mediated adhesion (Toyoshima and Nishida, 2007) can bias spindleorientation in cultured cells. Also, the Spire and Formin families ofactin nucleators have been shown to cooperate in the asymmetricpositioning of the meiotic spindle during oocyte division byestablishing a cortical actin network necessary for spindlemovement (Pfender et al., 2011). Finally, how do MT enzymes thatregulate growth and catastrophe dynamics contribute to +TIParchitecture, and how do MT dynamics regulate coupling tocortical complexes? It seems plausible that the geometry of the MTplus-ends relative to the cell cortex is vital for establishingproductive interactions that promote spindle movements (Su et al.,2012). We suggest that a significant role of +TIPs is to shape MTarchitecture dynamically to provide effective cortical couplingnecessary for spindle orientation, possibly through enhancement ofcortical force generators (Fig. 2D) (Kozlowski et al., 2007).Although a significant effort has been made to understand the D

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cortical cues involved in spindle orientation, further studies thataddress the role of the microtubule plus-end will help to elucidatethe molecular interplay involved in cortical coupling.

Bridging the gap between polarity and spindlepositioning: Pins-Dlg-Khc-73, an MT-capturingcomplexOne fundamental aspect of spindle orientation is the ability ofcortical cues to capture the spindle physically, probably as an initialstep in dictating its ultimate position. Our discussion of +TIPsabove demonstrates the variety of potential microtubule prey in thiscapture event. What then is the cortical bait that secures the spindleconnection? How are these cortex-microtubule interactionsestablished, maintained and regulated? What are the functionalconsequences of spindle capture on other aspects of the spindleorientation process? Static spindle-cortex connections could allowfor proper localization and/or activation of pathways that act insubsequent steps; for example, the microtubule-mediatedoffloading of force-generating molecules to the cortex (Markus andLee, 2011; Pecreaux et al., 2006). Below, we discuss the regulationof a recently characterized cortical spindle-capturing complex andpresent a model for how it cooperates with an evolutionarilyconserved force-generating complex.

As discussed earlier, the evolutionarily conserved polarity proteinPins regulates spindle orientation in a variety of cell types withindiverse organisms to achieve distinct biological outcomes (David etal., 2005; Du et al., 2001; Schaefer et al., 2001). Recently, weidentified a ‘microtubule capture’ pathway in Drosophila thatinvolves a Pins-Dlg-Khc-73 complex (Fig. 3A). Discs large (Dlg)binds directly to a short, linear motif in the ‘Linker’ domain of Pinsand is required for proper spindle orientation in both an ‘inducedcell polarity’ S2 cell system and neuroblasts (Johnston et al., 2009;Siegrist and Doe, 2005). Interestingly, this interaction is dependentupon phosphorylation of Pins by the mitotic, centrosomal kinaseAurora-A (Aurka in mammals; Aur in Drosophila), as is Pins-mediated spindle orientation (Johnston et al., 2012; Johnston et al.,2009; Lee et al., 2006a). In this model, Dlg associates with the cellcortex and localizes with polarized Pins (Siegrist and Doe, 2005).Dlg then interacts with Khc-73, a kinesin motor protein that movestowards and localizes at microtubule plus-ends (Hanada et al., 2000;Huckaba et al., 2011; Siegrist and Doe, 2005); Dlg binding appearsto enhance Khc-73 activity in vitro (Yamada et al., 2007). Dlgbinding occurs through a unique domain of Khc-73, termed themaguk binding stalk (MBS). Thus, Dlg serves as an adaptor proteinthat links polarized Pins at the cell cortex to the microtubule plus-end motor, Khc-73, and loss of any of these three componentsresults in spindle orientation defects (Fig. 3A). Interestingly, boththe Pins Linker and Khc-73 MBS domains bind to the guanylatekinase (GUK) domain of Dlg (Johnston et al., 2009; Siegrist andDoe, 2005), suggesting that they bind at distinct sites within theGUK domain. The structural basis for phosphorylation-dependentPins-Dlg complex formation has recently been detailed (Johnston etal., 2012; Zhu et al., 2011a) but a structural analysis of the MBSdomain alone or in complex with Dlg is lacking. It remains to bedetermined whether Khc-73 motor activity per se is required forspindle orientation, although misexpression of the MBS domainalone functions as a dominant negative, suggesting that otherdomains are indeed required (Johnston et al., 2009; Siegrist andDoe, 2005).

Using the induced polarity assay in Drosophila S2 cells, wefound that although full-length Pins could robustly orient themitotic spindle, the Linker domain alone was sufficient for a

‘partial orientation’ phenotype. We found that this intermediateactivity of the isolated Linker domain typically oriented spindlesprecisely at the distal edge of the cortical Pins crescent (as opposedto the center of the crescent with full-length Pins). Live-cellimaging experiments revealed that mitotic spindles with polescontacting non-Pins-expressing regions of the cell cortex rotatedslowly before becoming secured at the edge of the Linker crescent(Johnston et al., 2009). These results suggest that the Pins-Dlg-Khc-73 complex functions to capture the mitotic spindle staticallyby establishing a physical connection between cortical polarity andmicrotubule plus-ends (Fig. 3A, Fig. 4).

Whether such a capture mechanism occurs with other polarityproteins remains to be determined; the plethora of microtubuleplus-end binding proteins discussed above suggests this mayindeed be a widespread component of spindle orientation. Alsounclear is the precise function of this spindle-capturing activity.Does static spindle capture allow for activation of additionalpathways at specific sites? Are other regulators localized oroffloaded to the cortex more efficiently from the astralmicrotubules of a stationary spindle? Do cortical force generatorsact with higher fidelity on a ‘captured’ spindle? Alternatively, thecapture of microtubules at the edge of a cortical polarity complexmay be sufficient for spindle orientation in certain cellular contexts.

Linking polarity to spindle force generation: therole of the Mud-dynein complexPerhaps one of the most well characterized effectors in spindleorientation pathways is the Mud-dynein complex (Figs 3, 4)(Gönczy, 2008; Siller and Doe, 2009). Drosophila Mud is a large(>2000 amino acid) coiled-coil protein originally identified as aregulator of neuroblast proliferation (Guan et al., 2000). Mudshares overall weak sequence homology with the C. elegans andmammalian functional orthologs LIN-5 and NuMA, respectively(Siller et al., 2006). However, these proteins share a relativelyhigher conservation in a small C-terminal domain termed the Pins-binding domain (PBD). The PBD directly interacts with the Pinstetratricopeptide repeat (TPR) domain, and this interaction requiresprior Gα protein-mediated relief of an autoinhibitory conformationbetween the TPR and GoLoco domains of Pins (Du and Macara,2004; Nipper et al., 2007). This interaction recruits Mud to the Pinscortical crescent, thereby polarizing Mud localization (Fig. 3A).Notably, Mud also localizes to spindle poles and microtubules,independent of Pins, where it functions in the establishment andproper maintenance of focused microtubules (Bowman et al., 2006;Izumi et al., 2006; Silk et al., 2009; Siller et al., 2006). Additionalstudies have shown that the scaffold protein, Canoe (Cno),promotes formation of the cortical Pins-Mud complex throughseveral small GTPases (e.g. Ran) and is important for properspindle orientation (Fig. 3A) (Speicher et al., 2008; Wee et al.,2011).

The function of Mud in Pins-mediated spindle orientationappears to be that of a cortex-to-microtubule adaptor protein,analogous to the role played by Dlg (Fig. 4). Mud associates withcytoplasmic dynein, which serves as the sole minus-end-directedmicrotubule motor protein during mitosis. Dynein is a large proteincomplex consisting of core light, intermediate and heavy chains,the latter of which possesses the ATPase activity necessary formovement along microtubules. The non-catalytic subunits serve asbinding sites for additional regulatory proteins, some of which areessential for dynein activity and, thus, spindle orientation (e.g.p150-dynactin and LIS-1) (Kardon and Vale, 2009; Siller and Doe,2008). Precisely how Mud interacts with dynein remains unclear,

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although recent evidence suggests that Mud associates with thedynein light chain subunit, Cut up (Ctp), which is required forcentrosomal localization of Mud and spindle orientation inneuroblasts (Fig. 3A) (Wang et al., 2011). Recent evidence alsosuggests that a cortically localized component of dynein, viaspecific interaction with Mud, is required for proper spindleorientation (Kotak et al., 2012). Thus, Mud facilitates a linkbetween the dynein motor complex and cortically polarized Pins.

In contrast to the ‘spindle capture’ mechanism proposed for Khc-73, dynein is thought to provide the ‘force generation’ required forrapid spindle repositioning (Gönczy, 2008). The minus-enddirectional movement of dynein along astral microtubules (towardsthe spindle pole) results in net pulling forces on the spindle towardscortical Pins tethered by Mud (Gusnowski and Srayko, 2011)(Fig. 3A). Spindle-severing experiments in C. elegans embryoshave established a role for the Pins-Mud-dynein complex ingenerating the force necessary for spindle positioning (Colombo et

al., 2003; Nguyen-Ngoc et al., 2007). Additionally, the polarizedlocalization of the Pins-Mud complex results in unequal pullingforces at one spindle pole, which probably contributes to theasymmetry in spindle morphology and subsequent size of daughtercells (Bowman et al., 2006; Izumi et al., 2006; Siller et al., 2006).

The requirement of the Mud-dynein complex for proper spindlepositioning has been firmly established in several systems.However, with our ‘induced polarity’ assay, we found that thiscomplex was not sufficient to regulate spindle orientation inisolation (Johnston et al., 2009). Instead, Mud-dynein functionedsynergistically with Dlg-Khc-73 downstream of Pins. Whetherdirect crosstalk exists between these two complexes, or if theyperform otherwise independent functions that diverge below Pins,remains to be determined. Kinesin family members play animportant role in plus-end delivery of various cargo proteins(Hirokawa et al., 2009), suggesting a model in which Khc-73 aidsin localization of Pins-Mud pathway components. Live-cellanalysis revealed that the PinsTPR-Mud-dynein component wasrequired the for rapid, dynamic spindle movement necessary forrobust orientation, further strengthening the model of Mud-dyneincomplex-mediated force generation. An important question forfuture investigation will be how the minus-end Mud-dyneinpathway synergizes with that of the plus-end Dlg/Khc-73.Interestingly, cooperative involvement between opposite end-directed motor protein pathways has also been seen in meioticspindle positioning (Ellefson and McNally, 2009).

Recently, the Mud-dynein complex was shown to regulatespindle orientation mediated through the Wnt-Frizzled (Fz) effectorDishevelled (Dsh) in both Drosophila and zebrafish (Fig. 3B)(Ségalen et al., 2010). The Fz-Dsh complex had long been knownto influence planar spindle orientation in the pI division ofDrosophila SOPs; however, a bona fide downstream pathway wasunknown (Bellaïche et al., 2001; Gho and Schweisguth, 1998).Both genetic and biochemical evidence demonstrated that Mud actsdirectly downstream of the Dishevelled-Egl10-Pleckstrin (DEP)

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Fig. 3. Molecular models for Pins- and Dsh-mediated spindleorientation. (A) The Cdc42-Par6-aPKC-Baz polarity complex associateswith Pins through the adapter protein Insc. Pins cortical association ismediated by direct binding between three consecutive GoLoco domainsand the heterotrimeric G-protein Gαi. Pins serves as the ‘hub’ for twodistinct spindle orientation pathways in Drosophila neuroblasts. First, Pins(via its TPR domain, not shown) binds directly to Mud, which associateswith cytoplasmic dynein through Ctp. Lis-1 functions as an activator ofdynein microtubule motor activity. The scaffold protein Cno is requiredfor maintenance of the apical Pins-Mud complex. The dynein-dynactincomplex moves processively towards MT minus-ends (arrow) andgenerates pulling forces on the mitotic spindle. Second, the Pins linkerdomain is phosphorylated (yellow circle) by Aurora-A kinase. Thisphosphorylation allows direct binding with Dlg, which then associateswith the kinesin motor protein Khc-73; the plus-end binding capacity ofKhc-73 is thought to provide an MT capture or attachment mechanism.Together, the MT-capture and force-generation pathways elicit robustspindle alignment. (B) The Frizzled receptor (Fz, blue) is planar polarizedin many cell types. Following Wnt activation, Fz recruits the cytoplasmicscaffold protein Dishevelled (Dsh, orange) to the cortex via its PDZdomain. The Fz-Dsh complex regulates spindle orientation in Drosophilasensory organ precursor cells through association (via the Dsh DEPdomain) with Mud. Similar to Pins, Mud associates with the dynein motorcomplex. Whether the Dsh-Mud pathway is sufficient for spindleorientation or if additional pathways (black box) are required remains tobe investigated.

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domain of Dsh (Fig. 3B). Although this model is analogous to thatof Mud acting downstream of the Pins TPR domain, the DEP andTPR domains share no similarities in primary sequence or tertiarystructure. These results, therefore, highlight a striking example ofconvergent evolution of a shared signaling pathway downstream ofdivergent upstream activators. Whereas the minimal TPR-bindingpeptide has been identified in Mud and its structure determined(Smith and Prehoda, 2011; Zhu et al., 2011b), the precise molecularbasis for the Dsh-Mud complex remains to be determined. Whetheradditional pathways act downstream of Dsh is unclear, and acomplete molecular model for Dsh-mediated spindle orientationremains to be described (Fig. 3B).

Despite ample research establishing an important role for theMud-dynein complex in spindle orientation, many questionsremain regarding its molecular function. How does Mud bindingaffect dynein activity? Structural and biophysical data arebeginning to illuminate the conformational changes dynein mightundergo to achieve its movement along microtubules (Carter et al.,2011; Redwine et al., 2012). Does Mud actively alter thesestructural transitions to enhance dynein function? How is synergyachieved between Mud-dynein and Dlg-Khc-73 complexesdownstream of Pins? Despite diverse sequence determinants forbinding, do Pins and Dsh regulate Mud-dynein function in similar

ways? Does the Mud-dynein pathway function together with asecondary pathway in Dsh-mediated spindle orientation?

Phosphoregulation of spindle orientation: anassortment of kinases regulates spindleorientationIntense efforts have recently been focused on identifying additionalregulators of spindle orientation. Notably, a network of proteinkinases has now been shown to influence spindle positioning invarious systems. Although an in depth discussion of each kinase isbeyond the scope of this Review, a cursory examination iswarranted and highlights the level of complexity to which thisbiological process has evolved.

As discussed above, APC localizes at plus-ends and regulatesMT stability and spindle orientation (Fleming et al., 2009; Wen etal., 2004; Yamashita et al., 2003). In early Drosophila embryos, thekinase Akt localizes to the cell cortex and is necessary for corticallocalization of APC, and reduction of Akt results in impropercentrosome positioning, bent mitotic spindles, and spindlemisorientation in epithelial cells (Buttrick et al., 2008). The p21-activated kinase (PAK) regulates astral microtubule formation andcontrols mitotic spindle orientation and cortical anchoring, possiblyby regulating localization of the dynein-dynactin complex

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Fig. 4. A summary of the mechanisms that contribute to spindle orientation. Cortical spindle orientation cues, such as Pins and Dsh (dark blue),polarize to specific regions of the cell cortex. Cortical adapters, such as Dlg and Mud/NuMA (light blue), are then recruited by direct interactions withcortical cues. Polarized cortical cues and adapters interface with spindle microtubules through interactions with various plus-end localized microtubule-binding proteins, including motor proteins (e.g. Khc-73 and Dynein; red) and additional regulatory complexes (e.g. EB1-APC; green). These elementsprovide microtubule capture and forge generation effects that coordinate spindle positioning. Additional plus-end binding proteins aid in microtubuledynamics, such as polymerases (e.g. XMAP215, dark green) and depolymerases (e.g. MCAK, light green), which are necessary for proper spindleassembly and function. An assortment of protein kinases (yellow) have been shown to regulate spindle orientation through various mechanisms,including regulation of microtubule architecture (e.g. LKB and c-Src), regulation of cortical cue-adapter complexes (e.g. ABL1 and AurA) and localizationof microtubule regulators such as Ran (gray) (e.g. PAK).

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(Bompard et al., 2013). PAK-mediated phosphorylation of the RanGTPase promotes active Ran at the centrosome (Bompard et al.,2010). Interestingly, Ran activation has been shown to promotecortical Mud localization in Drosophila (Speicher et al., 2008; Weeet al., 2011). Thus, PAK phosphorylation may act to initiate Ran-mediated formation of the Pins-Mud spindle orientation complex.

The energy-regulated AMP-activated kinase (AMPK; SNF1A inDrosophila; PRKAA1 in mammals) localizes to spindle polesduring mitosis, and a recent study of human cell culture systemsdemonstrated that loss of AMPK results in astral microtubuleabnormalities and spindle misorientation (Thaiparambil et al.,2012). This study identified the myosin regulatory light chain(MRLC) as an essential AMPK target, although howphosphorylated MRLC regulates spindle orientation remainsunknown. Additionally, the spindle orientation activity of AMPKis dependent upon activation by the upstream kinase LKB1(Thaiparambil et al., 2012; Wei et al., 2012). Cell culture studieshave also demonstrated a role for the tyrosine kinase c-Src (alsoknown as Csk) in spindle orientation. Spindle misorientation isseen in early prometaphase upon loss of c-Src, which localizes tospindle poles; however, no apparent c-Src target has been identified(Nakayama et al., 2012). Cells deficient in c-Src demonstrateseverely reduced astral microtubules, suggesting the role of c-Srcis to promote spindle pole development. Notably, an earlier studyin C. elegans showed that c-Src (CSK-1) is necessary for properspindle rotation in the EMS cell division and proposed that thekinase functions downstream of Wnt signaling, which is necessaryfor proper endoderm specification (Bei et al., 2002).

Using a HeLa cell model, and translating findings in vivo tomouse epidermis, Matsumura and colleagues recently performed akinase-targeted RNAi screen for regulators of spindle orientationand identified the Abelson kinase (ABL1) (Matsumura et al.,2012). Loss of spindle orientation relative to the cell-substrateadhesion plane resulted from delocalization of the human Pinshomolog LGN (also known as GPSM2) from polarized to uniformcortical. Moreover, ABL1 phosphorylation of the Mud homologNuMA was necessary for maintenance of the LGN-NuMA corticalcomplex. It remains to be determined precisely how ABL1phosphorylation regulates localization of the LGN-NuMAcomplex. A similar delocalization of LGN was recently shown inan epithelial cell system upon loss of aPKC (Hao et al., 2010).aPKC phosphorylates LGN at the apical surface, which promotes14-3-3 binding and removal of LGN from this region of the cellcortex. In the absence of aPKC-mediated phosphorylation, LGNaccumulates at the apical cortex and promotes the loss of planarspindle orientation. Interestingly, this phosphorylation occurs at theconserved Aurora-A phosphorylation site previously shown topromote the Pins-Dlg pathway (Johnston et al., 2012; Johnston etal., 2009). This suggests that diverse cell types might use differentkinase-dependent pathways phosphorylating the same position toachieve unique spindle orientation outcomes.

Collectively, these studies have revealed an important role forphosphoregulation of spindle orientation pathways (Fig. 4). Acommon theme for kinase function appears to be regulatingsubcellular localization of specific spindle orientation components.In some cases, phosphorylation promotes cortical localization,whereas it results in cortical removal in other cases. Manyunanswered questions await further research in this area. What arethe molecular mechanisms involved in phosphorylation-dependentlocalization patterns? How does phosphorylation affect directprotein-protein interactions underlying the macromolecularcomplexes involved in spindle orientation? What specific residues

are phosphorylated in each of these components? What are thespatiotemporal constraints of phosphoregulation? Finally, what arethe phosphatases that direct dephosphorylation and how are theyregulated throughout the cell cycle?

ConclusionsThe ability to orient cell division allows for the generation ofcellular diversity and for regulated spatial placement of daughtercells within tissues. Nature has evolved sophisticated signalingpathways that allow cortical cues to communicate with the mitoticspindle to influence its position prior to cell division (Fig. 4). Themicrotubule plus-end is a node for the localization of diverse +TIPsthat serve as spindle-capture prey. Whether autonomously localizedproteins, such as XMAP215 and CLIP-170, or cargo of theessential +TIP EB1, such as APC, these assorted members regulatemicrotubule organization and aid in the cortical connection of thespindle. The cortex, by contrast, contains orientation cues, such asPins and Dsh, that organize protein complexes to function asspindle-capturing bait as well as force generators for the rapidspindle motions required for repositioning during cell division. Inthis Review, we have summarized many of the seminal findingsdescribing the molecular basis for spindle orientation. Althoughsignificant progress has been made on this front, it is apparent thatthe surface has merely been scratched. What additional +TIPsmight serve as spindle-capturing prey? How do microtubuledynamics and the regulatory proteins involved relate to spindlecapture and force generation? What other cortical complexes serveas spindle positioning cues, and in what cell types do thesefunction? Is dynein the lone force generator involved in spindleorientation, and how is the dynein complex regulated at themolecular level? How do divergent spindle capturing eventscooperate with the conserved force-generating complex? Finally,does the association between spindle orientation defects and humandisease play a causal role, and can spindle orientation regulatorsrepresent potential therapeutic targets? We excitedly await furtheranalysis of this fascinating biological process.

AcknowledgementsThe authors would like to thank Dr Chris Q. Doe for careful reading of themanuscript and insightful comments.

FundingMuch of the authors’ personal work discussed in this Review was funded by aNational Institutes of Health Training grant [M.S.L.] and a Damon RunyonCancer Research Foundation fellowship award [C.A.J.]. Deposited in PMC forrelease after 12 months.

Competing interests statementThe authors declare no competing financial interests.

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